WO1997028183A1

WO1997028183A1 - Peptide inhibitors of hematopoietic cell proliferation

Info

Publication number: WO1997028183A1
Application number: PCT/IB1997/000221
Authority: WO
Inventors: Josiane Thierry; Joanna Wdzieczak-Bakala; Pierre Potier; Maryse Lenfant; Catherine Grillon
Original assignee: Societe De Conseils De Recherches Et D'applications Scientifiques S.A. (S.C.R.A.S.)
Priority date: 1996-02-01
Filing date: 1997-01-31
Publication date: 1997-08-07
Also published as: AU1807597A; EP0877753A1; IL125509A0; CA2244673A1; JP2002515864A; AU721261B2; PL328081A1

Abstract

The invention features derivatives of the tetrapeptide Ser-Asp-Lys-Pro and the use thereof to inhibit the proliferation of hematopoietic cells.

Description

PEPTIDE INHIBITORS OF HEMATOPOIETIC CELL PROLIFERATION

Background of the Invention The tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) was originally isolated from fetal calf bone marrow. Lenfant, et al., Proc. Natl. Acad. Sci. USA 86:779-782 (1989) . AcSDKP is a negative regulator of hematopoietic stem cell proliferation, preventing stem cell recruitment into the S-phase. Frindel, et al., Exp. Hematol. 5:74-76 (1977) . AcSDKP appears to exert this function by blocking the action of stem cell-specific proliferation stimulators. Robinson, et al., Cell Proliferation 25:623-32 (1992). Phase-specific anticancer drugs (e.g., Ara-C or cisplatin) or radiation act on cells committed to proliferation, irrespective of whether the cell is malignant. Thus, administration of AcSDKP in conjunction with cytotoxic therapy, protect normal hematopoietic progenitor cells in the quiescent state.

Summary of the Invention In one aspect, the invention features compounds of the formula:

wherein

A_χ is the identifying group of the D- or L- isomer of Ser; A₂ is the identifying group of the D- or L- isomer of Asp or Glu;

A₃ is the identifying group of the D- or L- isomer of Lys, Arg, or Orn; A is the D- or L- isomer of Pro; R_x is H, Cχ-i₂ alkyl, C₇_₂₀ arylalkyl, R₇CO, or R₇OC(0) , where R₇ is C_1-12 alkyl, C₇_₂o arylalkyl, or C^^ alkyl or C₇_₂₀ arylalkyl substituted, e.g., one to three times, with OH, C0₂H, or NH₂;

R₂ is H, C_1-12 alkyl, or C₇_₂₀ arylalkyl; each of R₃ and R₄, independently, is CO-NH, CH₂- NH, CH₂-S, CH₂-0, CO-CH₂, CH₂~CO, 0-CH₂-CH₂; R₅ is CO or CH ; and R₆ is OH, NH₂, C_χ_₁₂ alkoxy, or NH-Y-CH₂-Z, where Y is a branched or straight chain C_1-12 hydrocarbon, e.g., branched or straight chain, moiety and Z is H, OH, C0₂H, or CONH₂; provided that if R₆ is OH, R₃ is CO-NH, and R₄ is CO-NH, then R₅ is CH₂; or a pharmaceutically acceptable salt thereof.

Examples of compounds of the invention are the following:

CH₃CO-Ser-Ψ(CH₂NH)-Asp-Lys-Pro-OH (Analog 1) ; CH₃CO-Ser-Asp-Ψ(CH₂NH)-Lys-Pro-OH (Analog 2) ; CH₃CO-Ser-Asp-Lys-ψ(CH₂N)Pro-OH (Analog 3); CH₃CO-Ser-Ψ(CH₂NH)-Asp-Lys-Pro-NH₂; CH₃CO-Ser-Asp-Ψ(CH₂NH)-Lys-Pro-NH₂; CH₃CO-Ser-Asp-Lys-Ψ(CH₂N)Pro-NH₂; H-Ser-Ψ(CH₂NH)-Asp-Lys-Pro-OH; H-Ser-Asp-Ψ(CH₂NH)-Lys-Pro-OH;

H-Ser-Asp-Lys-Ψ(CH₂N)-Pro-OH; HOOCCH₂CH₂CO-Ser-Ψ(CH₂NH)-Asp-Lys-Pro-OH; HOOCCH₂CH₂CO-Ser-Asp-Ψ(CH₂NH)-Lys-Pro-OH; HOOCCH₂CH₂CO-Ser-Asp-Lys-Ψ(CH₂N)Pro-OH; H-Ser-Ψ(CH₂NH)-Asp-Lys-Pro-NH₂; H-Ser-Asp-Ψ(CH₂NH)-Lys-Pro-NH₂; H-Ser-Asp-Lys-Ψ(CH₂N)-Pro-NH₂; HOOCCH₂CH₂CO-Ser-Ψ(CH₂NH)-Asp-Lys-Pro-NH₂; HOOCCH₂CH₂CO-Ser-Asp-Ψ(CH₂NH)-Lys-Pro-NH₂; HOOCCH₂CH₂CO-Ser-Asp-Lys-Ψ(CH₂N)Pro-NH₂; CH₃CO-Ser-Asp-Lys-Pro-NH₂; H-Ser-Asp-Lys-Pro-NH₂; CH₃CO-Ser-Asp-Lys-Pro-NHCH₃; H-Ser-Asp-Lys-Pro-NHCH₃; HOOCCH₂CH₂CO-Ser-Asp-Lys-Pro-NHCH₃; and HOOCCH₂CH₂CO-Ser-Asp-Lys-Pro-NH_2< With the exception of the N-terminal amino acid and Pro, all abbreviations (e.g., Asp) of amino acids in this disclosure stand for the structure of -NH-CH(R)-CO-, wherein R is a side chain "identifying group" of an amino acid (e.g., CH₂0H for Ser, CH₂COOH for Asp, CH₂CH₂COOH for Glu, CH₂CH₂CH₂NHC(NH₂)NH₂ for Arg, (CH₂)₃NH₂ for Orn, and (CH₂)₄NH₂ for Lys). For the N-terminal amino acid, the abbreviation stands for the structure of =N-CH(R)-C0- or -NH-CH(R)-CO-, wherein R is the identifying group of the amino acid. Pro is the abbreviation of prolyl. By non-peptide bond or pseudopeptide bond is meant that, where the α-amino group of proline is not involved, the peptide CO-NH bond between two amino acid residues is replaced with a non- peptide bond, e.g., CH₂-NH, CH₂-S, CH₂-0, CO-CH₂, CH₂-C0, or CH₂-CH₂ (symbolized by Ψ(CH₂-NH) or the like); or that, where the α-amino group of proline is involved, the carbonyl group of the peptide bond is replaced with CH₂ (symbolized by Ψ(CH₂-N)). C_1-12 alkyl and C_x_₁₂ alkoxy may be straight chained or branched, e.g., methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, or isopropoxy. C₇_₂₀ arylalkyl may be straight chained or branched, e.g., benzyl, napthyl, or phenylethyl. The compounds of the present invention can be used to inhibit the proliferation of hematopoietic cells. The compounds of the invention can be used to protect hematopoietic cells (e.g., stem cells) during treatment with cytotoxic agents (e.g., chemotherapy) or radiation (e.g., radiotherapy). The compounds of the invention may be administered prior to the administration of the cytotoxic agent or radiation and continued through the duration of the cytotoxic treatment or radiation.

The compounds of this invention can be provided in the form of pharmaceutically acceptable salts.

Acceptable salts include, but are not limited to, acid addition salts of inorganic acids such as hydrochloride, sulfate, phosphate, diphosphate, hydrobromide, and nitrate; or salts of organic acids such as acetate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, palmoate, salicylate, oxalate, and stearate. Also within the scope of the present invention, where applicable, are salts formed from bases such as sodium or potassium hydroxide. For further examples of pharmaceutically acceptable salts see, "Berge et al.", J. Pharm. Sci. 66:1 (1977).

A therapeutically effective amount (e.g., an amount effective to reduce the proliferation of hematopoietic cells) of a compound of this invention and a pharmaceutically acceptable carrier substance (e.g., magnesium carbonate, lactose, or a phospholipid with which the therapeutic compound can form a micelle) together form a therapeutic composition (e.g., a pill, tablet, capsule, or liquid) for administration (e.g., orally, intravenously, transdermally, pulmonarily, vaginally, subcutaneously, nasally, ionphoretically, or intratracheally) to a subject in need of the compound. The pill, tablet, or capsule can be coated with a substance capable of protecting the composition from the gastric acid or intestinal enzymes in the subject's stomach for a period of time sufficient to allow the composition to pass undigested into the subject's small intestine. The therapeutic composition can also be in the form of a biodegradable or sustained release formulation for subcutaneous or intramuscular administration. See, e.g., U.S. Patents 3,773,919 and 4,767,628 and PCT Application No. WO 94/00148. Continuous administration can also be obtained using an implantable or external pump (e.g., INFUSAID™ pump) to administer the therapeutic composition.

The dose of a compound of the present invention for protecting hematopoietic cells varies depending upon the manner of administration, the age and the body weight of the subject, and the condition of the subject to be treated, and ultimately will be decided by the attending physician or veterinarian. Such an amount of the compound as determined by the attending physician or veterinarian is referred to herein as a "therapeutically effective amount." The compound of the present invention may also be administered with a cytotoxic agent or radiation. Examples of cytotoxic agents include, but are not limited to, daunorubicine, cyclophosphamide, taxol, 5-fluorouracil, dioxorubicine, cisplatin, methotrexate, cytosine, arabinoside, mitomycin C, prednisone, vindesine, carboplatinum, vincristine, or 3'-azido- 3'deoxythymidine (AZT) . The compound of the present invention may also be administered with an angiotensin converting enzyme (ACE) inhibitor. Examples of ACE inhibitors are listed in Jackson, et al., Renin and Angiotensin, in Goodman & Gillman's, The Pharmacological Basis of Therapeutics, 9th ed. , eds. Hardiman, et al. (McGraw Hill, 1996).

Also contemplated within the scope of this invention is a compound covered by the above generic formula for use in protection of hematopoietic cells during cytotoxic treatment, e.g., chemotherapy, viral treatment, or radiation treatment.

Other features and advantages of the present invention will be apparent from the detailed description and from the claims. Detailed Description of the Invention It is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference.

Synthesis The synthesis of short peptides are well examined in the art. The peptides of the invention were synthesized using the following general synthesis procedure.

All protected amino acids were purchased from Bachem (Bobendorf, Switzerland) , Calbiochem (San Diego, CA) , or Nova Biochem (La Jolla, CA) . Mass spectra were obtained using a mass spectrometer (MS50) using a xenon fast atom bombardment (FAB) gun using glycerol, thioglycerol, or nitrobenzyl alcohol as a matrix. Thin- layer chromatography (TLC) was performed on silica gel precoated plates (60F 254, Merck, Darmstadt, Germany). The following solvent systems were used: A) dichloromethane/methanol, 95/5; B) dichloromethane/methanol 9/1; C) ethyl acetate/heptane, 1/1; D) n-butanol/acetic acid/water, 4/1/1; and E) n- butanol/acetic acid/water/pyridine, 1/1/1/1. UV light, ninhydrin, and/or Pataki reagent were used for detection. Protected peptides were purified by chromatography on Merck silica gel 60 (40-60μm) columns. All reagents and solvents were of analytical grade and used as supplied except for tetrahydrofuran (THF) which was either distilled from sodium/benzophenone or filtered through a column of basic alumina immediately prior to use and dimethylformamide (DMF) which was distilled from ninhydrin under reduced pressure and stored over 4 angstrom molecular sieves. Protected peptides were characterized by their fast atom bombardment (FAB) or secondary ion mass spectra (SIMS) . High pressure liquid chromatography (HPLC) purifications were performed on a reverse phase Beckmann Ultrasphere C-18 column (5μ particle size, 10 x 250 mm; Beckman, Fullerton, CA) using either a gradient or an isocratic elution with a mixture of acetonitrile and water containing 0.1% trifluoroacetic acid (TFA) at 3 ml/ in flow rate. Elution was monitored by recording absorbance at 210 nm. Pure peptides were characterized by their FAB or SI mass spectrum. HPLC analysis for purity control was performed on a Novapak column C-18, 5μm (3.9 x 150 mm; Waters, Milford, MA) with a solvent system consisting of a binary system of water and acetonitrile containing 0.1% TFA at 1 ml/min flow rate with monitoring at 210 nm. The solvent program involved the following linear gradients: 1) 0% to 50% acetonitrile over 50 min, 2) 0% to 80% acetonitrile over 40 min. k values are reported in the two solvent systems.

The following is the description of the synthesis of N-Ac-Ser-Asp-ψ(CH₂NH)-Lys-Pro-OH (Analog 2). The abbreviations Ac, Z, Boc, t-But, and Bzl mean, respectively, acetyl, benzyloxycarbonyl, tert- butoxycarbonyl, tert-butyl, and benzyl.

(1) N-α-(Z)-N-ε-(Boc)-L-lysyl-L-proline-tert-butylester To a stirred solution of Z-Lys(Boc)-0H (2.66 g. 7 mmol) in THF (35 ml) , cooled to -15°C, was added N- methylmorpholine (0.77 ml, 7 mmol) followed by isobutylchloroformate (0.98 ml, 7 mmol) . The solution was stirred at -15°C for 5 min and then cooled to -20°C. Proline tert-butyl ester (1.32 g, 7.7 mmol), dissolved in DMF, was added. The temperature was maintained at -10°C for 1 h, and the solution was then allowed to warm up to room temperature. After stirring for 5 h, the reaction mixture was concentrated under reduced pressure. The residue was then dissolved in ethyl acetate (200 ml) and 5% citric acid (50 ml) . The aqueous layer was extracted with ethyl acetate (50 ml) . The pooled organic layers were washed with water, 5% sodium bicarbonate, and brine, dried over Na₂S0₄, and concentrated under reduced pressure to afford the protected dipeptide as an oil (3.6 g; yield: 96%).

(2) N-ε-(Boc)-L-lysyl-L-proline-tert-butylester

The oil from step (1) (1.08 g; 2 mmol) was dissolved in ethanol (40 ml) . 10% palladium on carbon catalyst

(0.120 g) was added, and the suspension was stirred for 4 hours 30 min under an atmosphere of hydrogen. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure (0.672 g; yield: 84%) .

(3) N-α-(Z)-9-(t-But)-L-aspartyl N,0-dimethyl hydroxamate

This compound was prepared and converted to the corresponding aldehyde as previously described by Martinez, et al., J. Med. Chem., 28:1878 (1985).

(4) N-α-(Z) -β-(O-t-But)-L-aspartyl-Y(CH₂NH)-N-ε-(Boc)-L- lysyl-L-proline-tert-butylester The aldehyde obtained in step (3) (2 mmol) was added to a solution of the dipeptide from step (2) (1 mmol) in methanol (MeOH) containing 1 percent of acetic acid (7 ml). Sodium cyanoborohydride (0.094 g) was added in portions over 30 min. After 2 hours 30 min, the reaction mixture was cooled on a ice-water bath and under stirring, and a cool saturated sodium bicarbonate solution was added at 0°C followed by ethyl acetate. The aqueous phase was extracted with ethyl acetate. The pooled organic layers were washed with water, dried over Na₂S0₄, and then concentrated under reduced pressure. The crude product was ehromatographed on silica gel using CH₂Cl₂/MeOH (99/1) and CH₂Cl₂/Me0H (98/2) as eluents to give the desired product (yield: 56%) .

(5) ,5-(0-t-But)-L-aspartyl-Ψ(CH₂NH)-N-ε-(t-Boc)-L-lysyl- L-proline-tert-butylester

The compound obtained in step (4) (0.5 mmol) was dissolved in ethanol (13 mL) . 10% Palladium on carbon catalyst (0.040 g) was added, and the suspension was stirred for 24 hours under an atmosphere of hydrogen.

Additional catalyst in water (1 ml) was added. After 24 hours, the catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure (0.277 g; yield: 100%) .

(6) N-α-(Z)-,3-(0-t-But)-L-seryl-3-(0-t-But)-L-aspartyl- Ψ(CH₂NH)-N-ε-(Boc)-L-lysyl-L-proline-tert-butylester

To a stirred solution of Z-(O-t-But)-Ser-OH (0.132 g, 0.45 mmol) in THF (2.5 ml) cooled to -15°C, was added N-methylmorpholine (0.050 ml, 0.45 mmol) followed by isobutylchloroformate (0.063 ml, 0.45 mmol). The solution was stirred at -15°C for 5 min then cooled to - 20°C. The tripeptide of step (5) dissolved in the minimum amount of dichloromethane was added. The temperature was maintained at -10°C for 1 hour, then allowed to warm up to room temperature. After stirring for 5 hours, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and 5% citric acid. The aqueous layer was extracted with ethyl acetate (AcOEt) (50 ml) . The pooled organic layers were washed with water, 5% sodium bicarbonate, and brine, and then dried over Na₂S0₄ and concentrated under reduced pressure to afford a white foam (0.351 g) . The crude product was ehromatographed on silica gel using AcOEt/Hexane (1/1) as an eluant (0.233 g; yield: 70%) .

(7) β-(O-t-But)-L-seryl-9-(O-t-But) -L-aspartyl-ψ(CH₂NH)- N-ε-(Boc)-L-lysyl-L-proline-tert-butylester The compound obtained in step (6) (0.2 mmol) was dissolved in 10% ethanol (4.4 ml). 10% Palladium on carbon catalyst (0.035 g) was added, and the suspension was stirred under an atmosphere of hydrogen overnight. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure (0.110 g) .

(8) N-α-(acetyl) -β-(O-t-But)-L-seryl-,3-(O-t-But)-L- aspartyl-Ψ(CH₂NH)-N-ε-(Boc)-L-lysyl-L-proline-tert- butyl ester

The amine of step (7) (0.110 g, 0.16 mmol) was dissolved in DMF (0.4 ml) and reacted with acetylimidazole (0.026 g, 0.24 mmol). After stirring for 3 hours, the reaction mixture was diluted with ethyl acetate. The organic phase was then washed with water and brine, dried over Na₂S0 , and concentrated under reduced pressure. The crude product (0.120 g) was ehromatographed on silica gel using AcOEt/MeOH(99/l) as an eluent (0.090 g; yield: 76%). (9) N-α-(acetyl)-L-seryl-L-aspartyl-Ψ(CH₂NH)-L-lysyl-L- proline-OH

The compound obtained in step (8) (0.086 g, 0.12 mmol) was dissolved in TFA/CH₂C1₂ (0.4 ml) . The solution was stirred for 2 hours 30 min at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was triturated with dry ether and dried under vacuum after removal of ether. Purification by HPLC on a C18 column using the following gradient solvent system: 0% at 3% acetonitrile over 10 min, 3% acetonitrile over 15 min with a flow rate of 3 ml/min (k'(l)= 8.17; k' (2)= 8) yielded the desired N-acetylated reduced tetrapeptide.

The following is the synthesis of CH₃CO-Ser-Asp-

Lys-Pro-NH₂. (Analog 9) (1) N-α-Benzyloxycarbonyl-N-ε-tert-Butoxycarbonyl-L- lysyl-L-proline-benzyl-ester

To a stirred solution of Z-Lys(Boc)-OH (1.54 g, 4 mmol) in THF (20 mL) , cooled to -150C, was added 0.5 mL (4 mmol) N-Methylmorpholine followed by 0.44 mL (4 mmol) isobutylchloroformate. The solution was stirred at -15fiC for 5 min and then cooled to -20BC. Benzylester proline hydrochloride (1.06 g; 4.4 mmol), in suspension in DMF (6 L) , was added followed by N-Methylmorpholine (0.48 mL, 4.4 mmol). The temperature was maintained below -loac for one hour and then allowed to warm up to room temperature. After 5 hours of stirring, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (120 mL) and 5% citric acid (60 mL) . The aqueous phase was extracted with ethyl acetate (60 mL) . The combined organic layers were washed with water, 5% sodium bicarbonate, and brine, then dried over Na₂S0₄, and concentrated under reduced pressure to afford the product as a syrup. The crude product was ehromatographed on silica gel using CH₂Cl₂/MeOH (99/1) as an eluant to give 1. Yield: 1.76 g (77%), R_f(CH₂Cl₂/MeOH, 98/2) - 0.22; R_f (AcOEt/ Heptane, 1/1) = 0.24, MS (FAB)m/z - 590 (MNa+) , 568 (MH+) , 512 (MH+-But) , 468 (MH+-BOC) , 434 MH+-Z) , 378 (MH+-Boc-Bzl) , 334 (MH+-BOC-Z) .

(2) N- -tert-Butoxycarbonyl-L-lysyl-L-proline

Product from step (1) (1 g; 1.75 mmol) was dissolved in 10% aqueous methanol (33 mL) . 10% palladium on carbon catalyst (0.200 g) was added, and the suspension was stirred under an atmosphere of hydrogen overnight. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure. Yield: 0.565 g (94%); R_f (n-Butanol/Acetic acid/Water, 4/1/1) = 0.55. MS (FAB) m/z = 366 (MNa+) , 344 (MH+) , 288 (MH+-BUt) , 244 (MH+-BOC) .

(3) N-α-Benzyloxycarbony1-0-O-tert-buty1-L-asparty1-N- - tert-Butoxycarbony1-L-lysy1-L-pro1ine

To a stirred solution of Z-L-Asp(O-t-But)-OH (0.323 g, 1 mmol) in THF (5 mL) , cooled to -152C, was added 0.11 mL (1 mmol) of N-Methylmorpholine, followed by 0.14 ml (1 mmol) isobutylchloroformate. The solution was stirred at -15SC for 5 minutes and then cooled to -202C. The product of step (2) was added in solution in DMF (2.5 mL) . After 5 hours stirring, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (50 mL) and 5% citric acid (25 mL) . The aqueous phase was extracted with ethyl acetate (25 mL) . The combined organic layers were washed with water and brine, then dried over Na₂S0₄, and concentrated, under reduced pressure, to afford a white foam. The crude product was ehromatographed on silica gel using CH₂Cl₂/MeOH/AcOH (97/3/0.5) as an eluant. Yield: 0.450 g (60%), R_f (CH₂Cl₂/MeOH/AcOH, 97/3/0.5) = 0.11 MS (FAB) m/z = 693 (M2Na+-H) , 671 (MNa+) , 615 (MNa+-But) , 549 (MH+- Boc) , 537 (MNa+-Z) , 515 (MNa+-But-Boc) , 493 (MH+-But- Boc) , 437 (MNa+-Boc-Z) .

(4) N-α-Benzyloxycarbonyl-ø-O-tert-butyl-L-aspartyl-N-ε- tert-Butoxycarbonyl-L-lysyl-L-proline amide

To a stirred solution of the product of step (3) (0.129 g, 0.2 mmol) in THF (5 mL) , cooled to -15CC, was added 0.022 mL (0.2 mmol) of N-Methylmorpholine followed by 0.028 mL (0.2 mmol) isobutylchloroformate. The solution was stirred at -15sc for 5 minutes, and then cooled to -202C. 0.2 mL of a cold 34% ammonia solution was added.

After one hour stirring, at a temperature below - 10BC, a further hour at a temperature below OSC, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and 5% citric acid. The aqueous phase was extracted with ethyl acetate. The combined organic layers were washed with water and brine, then dried over Na₂S0₄, and concentrated under reduced pressure to afford a white foam. The crude product was ehromatographed on silica gel using CH₂Cl₂/MeOH (95/5) as an eluant. Yield: 0.105 g (81%). R_f (CH₂Cl₂/MeOH, 95/5) = 0.24; R_f (AcOEt/MeOH, 99/1) = 0.45. MS (FAB) m/Z = 670 (MNa+) , 648 (MH+) , 614 (MNa+- But), 548 (MH+-BOC) , 534 (MH+-ProNH₂) , 514 (MH+-Z) , 492 (MH+-But-Boc) .

(5) /3-O-tert-butyl-L-aspartyl-N-ε-tert-Butoxycarbonyl-L- lysyl-L-proline amide

The product of step (4) (0.152 g; 0.23 mmol) was dissolved in methanol (6 mL) , 10% palladium in carbon catalyst (0.030 g) was added, and the suspension was stirred under an atmosphere of hydrogen for 2 hours. The catalyst was removed by filtration on a Celite pad, and the filtrate was concentrated under reduced pressure. Yield: 0.111 g (94%); R_f (CH₂Cl₂/MeOH, 95/5) = 0.08; R_f (CH₂Cl₂/MeOH, 9/1) = 0.35. MS (FAB) m/z = 536 (MNa+) , 514 (MH+) , 480 (MNa+-But) , 458 (MH+-But) , 414 (MH+-Boc) , 400(MH+-ProNH₂) .

(6) N-c.-Benzyloxycarbonyl-L-seryl-/3-0-tert-butyl-L- aspartyl-N-ε-tert-Butoxycarbonyl-L-lysyl-L-proline amide

To a stirred solution of Z-L-Ser-OH (0.045 g, 0.19 mmol) in THF (1 mL) , cooled to -15^βC, was added 0.021 mL (0.19 mmol) N-Methylmorpholine followed by 0.026 mL (0.19 mmol) isobutylchloroformate. The solution was stirred at -152C for 5 minutes and then cooled to -200C. The product of step (5) (0.105 g, 0.2 mmol) was added in solution in DMF (1 mL) .

After 5 hours stirring, the reaction mixture was concentrated under reduced pressure. The reaction mixture was dissolved in ethyl acetate (50 mL) and 5% citric acid (25 mL) . The aqueous phase was extracted with ethyl acetate (25 mL) . The combined organic layers were washed with water and brine, then dried over Na₂S0₄, and concentrated, under reduced pressure. The crude product was purified on a silica gel column using CH₂Cl₂/MeOH (94/6) as an eluant. Yield: 0.140 g (80%). R_f (CH₂Cl₂/MeOH, 95/5) = 0.18; R_f (CH₂Cl₂/MeOH, 9/1) = 0.43. MS (FAB) m/z = 757 (MNa+) , 735 (MH+) , 701 (MNa+- But) , 635 (MH+-BOC) , 601 (MH+-Z) , 579 (MH+-But-Boc) , 501 (MH+-BOC-Z) , 465 (MH+-Boc-But-ProNH₂) .

(7) N-α-acetyl-L-seryl-0-O-tert-buty1-L-asparty1-N-ε- tert-Butoxycarbonyl-L-lysyl-L-proline amide

The product of step (6) (0.080 g, 0.011 mmol) was dissolved in AcOEt (2 mL) . 10% palladium carbon catalyst (0.016 g) and acetylimidazole (0.014 g, 0.013 mmol) was added, and the suspension was stirred under an atmosphere of hydrogen overnight. The catalyst was removed by filtration on a Celite pad, and the filtrate was concentrated under reduced pressure. The crude product was purified on a gel column using CH₂Cl₂/MeOH (9/1) as an eluant. Yield: 0.050 g (71%); R_f (CH₂Cl₂/MeOH, 9/1) = 0.29. MS (FAB) m/z = 665 (MNa+) , 643 (MH+) , 609 (MNa+- But) , 543 (MH+-BOC) , 487 (MH+-But-Boc) , 373 (MH+-Boc-But- ProNH₂) .

(8) N-α-acety1-L-sery1-L-asparty1-L-lysy1-L-proline amide

The product of step (7) (0.039 g, 0.06 mmol) in solution in 200 μl trifluoroacetic acid, containing 20 μl of water, was stirred at room temperature for 95 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was triturated twice with dry ether. After removal of ether, the solid white residue was taken up in 1.5 ml water and lyophilized. The crude peptide was purified by HPLC on C-18 column (Beckman Ultrasphere ODS (10 x 250 mm)) using an elution consisting of two solvents (A: H₂O/0.1% TFA, B: acetonitrile 0.1% TFA; 100% to over 20 minutes; tR = 13 minutes) with a flow rate of 3 ml.min-1. The collected fraction was lyophilized and analyzed by HPLC on a Waters Nova-Pak C-18 column (Waters, Milford, MA), 4 μ, 80 λ (3.9 x 150 mm) with two different elution programs using the same solvent system as above and a 1 ml.min-1 flow rate, k = 8.4, 100 to 50% A over 50 min.; k = 7.1, 100 to 20% A over 40 min.; MS (FAB)m/z = 509 (MNa+) , 487 (MH+) . Other substitutions may similarly be added to the N-terminus of the peptide by similar methods known in the art. For example, N-α-(HOOCCH₂CH₂CO)-3-(O-t-But)-L-Ser-j3- (O-t-But)-L-aspartyl-Ψ(CH₂NH)-N-e-(Boc)-L-lysyl-L- proline-OH may be synthesized by mixing the amine of step (7) above dissolved in the minimum amount of CH₂C1₂ with a solution of succinic anhydride dissolved in THF. The reaction is stirred at room temperature and then the mixture is evaporated under reduced pressure. The residue is dissolved in AeOEt and washed with 5% citric acid, water, brine, and then dried over Na₂S0₄. The resulting compound may then be deprotected to yield the desired product.

Other peptides of the invention can be prepared in an analogous manner by a person of ordinary skill in the art.

Biological activity of AcSDKP analogues

The activity of the compounds of the invention was evaluated by their ability to inhibit the in vitro entry into S-phase of murine primitive hematopoietic cells:

"HPP-CFC". In order to trigger the quiescent stem cells into cycle, normal murine bone marrow cells (5 x 10⁶ cells/ml in Dulbecco's medium) were incubated with the same volume of either stimulatory medium (conditioned medium of bone marrow cells obtained from sublethally irradiated mice, 4.5 GY whole body X-l irradiated upon dose), or with Dulbecco's medium as control. Test compounds were added at the beginning of the incubation at a final concentration of 2 x 10~⁹ M. Incubations were performed in pair tubes at 37°C for 3 h. One hour before the end of the incubation, cells in S-phase were killed by adding cytosine arabinoside (Ara-C) at a final concentration of 25 μg/ml in the first set of tubes. Dulbecco's medium is added in the other tubes as control. Incubation with Ara-C leads to the death of cells which have been triggered into S-phase. Therefore, cells which have been prevented to cycle by the action of analogues will be insensitive to the phase-specific toxicity of Ara-C. Cells were washed twice prior to subsequent HPP- CFC assay.

HPP-CFC were studied using a bilayer semi-solid agar assay as described by Robinson, et al., Cell Prolif. 25:623-632, 1992. Two milliliters of Dulbecco's medium containing 20% horse serum, 10% conditioned medium from the WEHI 3B myelomonocytic leukaemic cell line (as a source of IL-3/multi-CSF) , 10% conditioned medium from L929 fibroblast cell line (as a source of M-CSF/CSF1) , 0.5% melted agar, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin were aliquoted into 55 mm diameter non-tissue culture grade plastic petri-dishes as the underlayer. Two milliliters of Dulbeccos's medium supplemented with 20% horse serum, 0.3% melted agar, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin containing 4 x 10⁴ bone marrow cells were then aliquoted over prepared underlayers. Quadruplicate cultures were incubated for 14 days at 37°C in a fully humidified atmosphere with 5% C0₂. Twelve hours before the end of the culture, 1 ml of a colorless 1 mg/ml 2-(4- iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) solution in saline was added, allowing the staining of viable cells by INT processing into a red derivative which precipitates inside cells. HPP-CFC derived macroscopic colonies were defined as those above 2 mm in diameter and scored. Table I lists the percent decrease of HPP-CFC derived macroscopic colonies entering the S-phase induced by the test compounds.

TABLE I

TEST PERCENT DECREASE OF COMPOUND HPP-CFC IN S-PHASE

Analog 1 61.2

Analog 2 67.6 Analog 3 70.8

Other Embodiments It is to be understood that while the invention has been described in conjunction with the detailed description thereof, that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the claims.

What is claimed is:

Claims

Claims 1. A compound of the formula:

wherein

A₂ is the identifying group of the D- or L- isomer of Ser;

A₂ is the identifying group of the D- or L- isomer of Asp or Glu;

A₃ is the identifying group of the D- or L- isomer of Lys, Arg, or Orn; A₄ is the D- or L- isomer of Pro;

R_j is H, CJ.-^ alkyl, C₇_₂₀ arylalkyl, R₇C0, or R₇OC(0), where R₇ is C_1-12 alkyl, C₇_₂₀ arylalkyl, or C₁_₁₂ alkyl or C₇_₂₀ arylalkyl substituted with OH, C0₂H, or NH₂; R₂ is H, C_1-12 alkyl, or C₇_₂₀ arylalkyl; each of R₃ and R₄, independently, is CO-NH, CH₂- NH, CH₂-S, CH₂-0, C0-CH₂, CH₂-C0, or CH₂-CH₂;

R₅ is CO or CH₂; and

R₆ is OH, NH₂, C_1-12 alkoxy, or NH-Y-CH₂-Z, where Y is a C_1-12 hydrocarbon moiety and Z is H, OH, C0₂H, or C0NH₂; provided that if R₆ is OH, R₃ is CO-NH, and R₄ is CO-NH, then R₅ is CH₂; or a pharmaceutically acceptable salt thereof.

2. A compound of claim 1, wherein A₂ is the identifying group of the D- or L-isomer of Asp and A₃ is the identifying group of the D- or L-isomer of Lys.

3. A compound of claim 2, wherein

A_j is the identifying group of L-Ser;

A₂ is the identifying group of L-Asp;

A₃ is the identifying group of L-Lys; and A is the identifying group of L-Pro.

4. A compound of claim 3, wherein each of R₃ and R₄, independently, is CO-NH or CH₂-NH.

5. A compound of claim 4, wherein R-_^ is H or R₇CO (where R₇ is C_1-12 alkyl or C_1-12 substituted with OH) and R₂ is H.

6. A compound of claim 5, further provided that if R₃ is CO-NH and R₄ is CO-NH, then R₅ is CH₂.

7. A compound of claim 6, wherein R_χ is H, CH₃C0, or HOOCCH₂CH₂CO and R₆ is OH or NH₂.

8. A compound of claim 1 of the formula:

CH₃CO-Ser-Ψ(CH₂NH)-Asp-Lys-Pro-OH; or a pharmaceutically acceptable salt thereof.

9. A compound of claim 1 of the formula: CH₃CO-Ser-Asp-Ψ(CH₂NH)-Lys-Pro-OH; or a pharmaceutically acceptable salt thereof.

10. A compound of claim 1 of the formula: CH₃CO-Ser-Asp-Lys-Ψ(CH₂N)Pro-OH;or a pharmaceutically acceptable salt thereof.

11. A compound of claim 1 of the formula: CH₃CO-Ser-Ψ(CH₂NH)-Asp-Lys-Pro-NH₂; CH₃CO-Ser-Asp-Ψ(CH₂NH)-Lys-Pro-NH₂; CH₃CO-Ser-Asp-Lys-Ψ(CH₂N)Pro-NH₂; H-Ser-Ψ(CH₂NH)-Asp-Lys-Pro-OH;

H-Ser-Asp-Ψ(CH₂NH)-Lys-Pro-OH;

H-Ser-Asp-Lys-Ψ(CH₂N)-Pro-OH;

HOOCCH₂CH₂CO-Ser-Ψ(CH₂NH)-Asp-Lys-Pro-OH;

HOOCCH₂CH₂CO-Ser-Asp-Ψ(CH₂NH)-Lys-Pro-OH; HOOCCH₂CH₂CO-Ser-Asp-Lys-Ψ(CH₂N)Pro-OH;

H-Ser-Ψ(CH₂NH)-Asp-Lys-Pro-NH₂, '

H-Ser-Asp-Ψ(CH₂NH)-Lys-Pro-NH₂;

H-Ser-Asp-Lys-Ψ(CH₂N)-Pro-NH₂;

HOOCCH₂CH₂CO-Ser-Ψ(CH₂NH)-Asp-Lys-Pro-NH₂; HOOCCH₂CH₂CO-Ser-Asp-Ψ(CH₂NH)-Lys-Pro-NH₂; and

HOOCCH₂CH₂CO-Ser-Asp-Lys-Ψ(CH₂N)Pro-NH₂; or a pharmaceutically acceptable salt thereof.

12. A compound of claim 5, wherein R₆ is NH₂ or NH-Y-CH₂-Z (where Y is a C_1-12 hydrocarbon moiety and Z is H) .

13 . A comppound of claim 12 , wherein R₃ and R₄ are CO-NH and R₅ is CO .

14 . A compound of cairn 13 , wherein R₂ is H, CH₃CO, or HOOCCH₂CH₂CO.

15. A compound of claim 14 of the formula :

CH₃CO-Ser-Asp-Lys-Pro-NH₂ ; or a pharmaceutically acceptable salt thereof .

16. A compound of claim 14, of the formula: H-Ser-Asp-Lys-Pro-NH₂; CH₃CO-Ser-Asp-Lys-Pro-NHCH₃; H-Ser-Asp-Lys-Pro-NHCH₃; HOOCCH₂CH₂CO-Ser-Asp-Lys-Pro-NHCH₃; and

HOOCCH₂CH₂CO-Ser-Asp-Lys-Pro-NH₂; or a pharmaceutically acceptable salt thereof.

17. A method of protecting hematopoietic cells in a subject undergoing chemotherapy or radiotherapy, said method comprising administering to said subject a therapeutically effective amount of a compound of claim 1, said amount being effective to reduce the proliferation of hematopoietic cells during said chemotherapy or radiotherapy.

18. A method of claim 17, wherein said compound is of the formula:

CH₃C0-Ser-Ψ(CH₂NH)-Asp-Lys-Pro-OH; CH₃CO-Ser-Asp-Ψ(CH₂NH)-Lys-Pro-OH; CH₃CO-Ser-Asp-Lys-Ψ(CH₂NH)-Pro-OH; CH₃CO-Ser-Asp-Lys-Pro-NH₂; or a pharmaceutically acceptable salt thereof.

19. A method of inhibiting the proliferation of hematopoietic cells in a patient, said method comprising administering to said patient a compound of claim 1.

20. A method of claim 19, wherein said compound is of the formula: